Viral nucleotide sequences

ABSTRACT

Various genes of herpes virus of turkeys (HVT) Marek&#39;s disease virus (MDV) and infectious laryngotracheitis virus (ILTV) have been identified as non-essential regions (and candidates for insertion sites for foreign genes) and/or as antigen-encoding regions. The former include the HVT homologue of the HSV (herpes simplex virus) gC gene, the TK (thymidine kinase) region of MDV or ILTV, ORF3 of ILTV (as defined herein), the ribonucleotide reductase (large subunit) gene of ILTV, MDV or HVT and the ribonucleotide reductase (small subunit) gene of MDV. The antigen-encoding regions include the HVT homologues of the HSV gB, gC and gH genes, the ILTV homologue of HSV gB, ORF2 of ILTV, and the HVT homologue of the HSV-1 immediate early genes IE-175 and IE-68. Manipulation of these genes allows vaccines to be prepared comprising attenuated virus or virus carrying heterologous antigen-encoding sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.09/133,409, filed Aug. 13, 1998, now U.S. Pat. No. 6,204,045, which is adivisional application of U.S. Ser. No. 08/125,039, filed Sep. 22, 1993,now U.S. Pat. No. 5,906,821, which is a continuation of U.S. Ser. No.07/669,391, filed Apr. 29, 1991, now abandoned, which is a nationalstage entry of PCT application PCT/GB89/01075, filed Sep. 13, 1989, andUnited Kingdom application Serial No. GB 8821441.6 filed Sep. 13, 1988.

The present invention relates to viral nucleotide sequences which may bemanipulated to provide vaccines against disease.

BACKGROUND AND DESCRIPTION OF PRIOR ART

Herpesviruses are large double stranded DNA viruses consisting of anicosahedral capsid surrounded by an envelope. The group has beenclassified as alpha, beta and gammaherpesviruses on the basis of genomestructure and biological properties [Roizman, B et al (1981)Intervirology 16, 201-217]. Avian herpes viruses include Marek's DiseaseVirus (MDV) (a gammaherpesvirus) which causes a lymphomatous disease ofconsiderable economic importance in chickens [reviewed in Payne, L. N.(ed) Marek's Disease (1985), Martinus Nijhoff Publishing, Boston] andInfectious Laryngotracheitis-Virus (ILTV) (an alphaherpesvirus) whichcauses an acute upper respiratory tract infection in chickens resultingin mortality and loss of egg production.

A recent unexpected finding in out laboratory is that there issufficient amino acid homology between MDV, ILTV and mammalianherpesviruses, particularly varicella zoster (VZV) and Herpes SimplexVirus (HSV) to allow identification of numerous conserved genes. Theseinclude the MDV and Herpesvirus of Turkeys (HVT) homologues ofglycoproteins gB, gC and gH of HSV; the ILTV, MDV and HVT homologues ofTK and ribonucleotide reductase genes and the ILTV homologue of gB andgenes 34 and 35 of VZV (Buckmaster, A et al, (1988) J. gen. Viral, 69,2033-2042.

Strains of MDV have been classified into three serotypes. Type 1comprises pathogenic strains and their attenuated derivatives. Type 2are a group of naturally-occurring non-pathogenic strains and type 3 isHVT. For more than a decade, vaccination with HVT has been remarkablyeffective in controlling Marek's disease.

However, in recent years, new strains of MDV have been isolated whichcause disease despite vaccination with HVT. Losses due to these ‘veryvirulent’ strains have occurred in parts of the U.S.A., Europe and theMiddle East. Although the degree of protection can be improved by usinga mixture of HVT, type 2 MDV and attenuated derivatives of very virulentstrains for vaccination, the results have been erratic,. Theseobservations and the fact that there are MDV type-specific epitopes thatare not shared by HVT or type 2 MDV have led us to the conclusion thatimproved vaccines might be constructed which are antigenically morerelated to MDV than existing vaccines. [Reviewed by Ross and Biggs inGoldman J. M. and Epstein M. A. (eds) Leukaemia and Lymphoma Research,Vaccine Intervention against Virus-Induced Tumour, p 13-31, Macmillan,1986].

Infectious laryngotracheitis is also a worldwide problem. Sporadicoutbreaks occur in which the severity of clinical symptoms variesconsiderably. Virus can persist in birds that have recovered and may beshed at intermittent intervals after recovery. An attenuated fieldstrain is currently used as a vaccine. However, it has retained somedegree of pathogenicity. Mortality due to the vaccine may reach 10% inyoung chicks.

A number of herpesvirus antigens have been shown to confer protectiveimmunity when expressed in a recombinant vaccinia virus. These includethe gB gene of HSV [Cantin E. M. et al (1987) Proc. Natl. Acad. Sci.U.S.A. 84, 5908-5912], gD of HSV [Paoletti, E. et al (1984) Proc. Natl.Acad. Sci. U.S.A. 81, 193-197] and gp50 of pseudorabies virus (PRV), ahomologue of HSV gD [Marchioli, C. C. et al (1987) J. Virol. 61,3977-3981]. Because of the absolute requirement of gB for viruspenetration and infectivity and because it is conserved amongherpes-viruses, gB and its homologues are important immunogens.Moreover, the presence of GOB at the surface of infected cells has beenshown to be an important target for humoral and cell-mediated immuneresponses [Blacklaws, B. A. et al J.gen. Virol. 68, 1103-1114 (1987);McLaughlin-Taylor, E. et al (1988) J. gen. Virol. 69, 1731-1734). Therecently described glycoprotein gH of HSV is also essential forinfectivity and may also be an important immunogen (Desai, P. J. et al(1988) J. gen. Virol. 69, 1147-1156]. It has also been shown that gIIIof pseudorabies virus (PRV), a homologue of gC, is a major target forneutralizing antibody and for cytotoxic T cells although it is anon-essential protein. Also of interest is the unexpected participationof immediate early proteins in T cell mediated cytotoxic reactions incells infected with cytomegalovirus (CMV) [Kozinowski U. H. et al (1987)J. Virol. 61, 2054-2058]. Similar antigens could play an important rolein the rejection of latently infected and transformed lymphocytes inMarek's disease since immediate early RNA transcripts have been detectedin lymphoblastoid cell lines established from Marek's disease tumours.

Although many recombinant vaccines have been constructed using thepoxvirus vaccinia as a vector, there are also reports of the use ofherpesviruses as vectors for the expression of foreign genes. Thushepatitis antigen has been expressed in HSV [Shih, M. F. et al (1984)Proc. Natl. Acad. Sci. U.S.A. 81, 5867-5870] and human tissueplasminogen activator has been expressed in PRV (Thomsen, D. R. et al(1987) Gene 57, 261-265. In both cases, foreign genes were inserted incloned fragments of non-essential herpes genes which were thenintroduced into the virus vector by homologous recombination. Thehepatitis virus gene was fused to a herpesvirus promoter and therecombinant DNA was inserted within the TK gene of HSV. Homologousrecombination following co-transfection of the recombinant DNA andwild-type HSV DNA resulted in TK− virus clones that expressed thehepatitis antigen.

In the case of PRV, the gX gene mapping in U_(s) was used as the sitefor insertion of the foreign gene. The strategy used involved insertionof the TK gene of HSV in the gX gene of a PRV mutant that had a defectin its TK gene resulting in a TK positive virus. The human tissueplasminogen activator gene was then inserted within a cloned fragementof HSV TK and the recombinant was introduced into the PRV mutant byhomologous recombination. TK− virus was selected which expressed thehuman gene (Thomsen et al as above). Similarly, VZV has been used as avector [Lowe et al (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3896-3900].

Several herpesvirus genes have also been shown to be associated withvirulence and to be non-essential for growth in vitro. These include theTK genes of HSV (Jamieson, A. T. et al (1974) J. gen. Virol. 24,465-480; Field, H. and Wildy, P., (1987) J. Hygiene (Cambridge) 81,267-2771 and of PRV. Indeed it has long been known that PRV is readilyattenuated by deletion of TK activity [Tatarov, G. (1968) Zentralbl.Vet. Med 15B, 848-853]. Furthermore, attenuation of the Bartha strain ofPRV has been attributed to a defect in gI, a non-essential structuralglycoprotein mapping in U_(s) [Mettenleiter, T. et al (1987) J. Virol.61, 4030-4032].

Genes of HSV mapping in the internal repeat region (TRS) flanking thelong unique sequence have also been associated with pathogenicity[Rosen, A. et al (1986) Virus Research 5, 157-175; Thompson, R. L. et al(1983) Virology 131, 180-192]. Several additional genes of HSV have beenshown to be non-essential for growth in vitro although it is not knownwhether they are associated smith virulence. These include UL24(Sanders, P. G., (1982), J. gen. Virol. 63, 277-295, large subunit ofribonucleotide reductase (Goldstein D. J. and Weller, S. K. (1988) J.

Virol. 62, 196-205), gC (Draper K. G. et al (1984) J.

Virol. 51, 578-585), dUTPase (Fisher, F. B. & Preston, V. G. (1986)Virology 148, 190-197), and U_(t)55 and U_(t) 56 (MacLean, A. R. &Brown, S. M. (1987) J. gen. Virol. 68, 1339-1350).

Moreover there is evidence that several genes of HSV mapping in U_(s)are also non-essential for growth in vitro [Weber, P. C. et al (1987)Science 236, 576-579].

SUMMARY OF THE INVENTION

One aspect of the present invention provides a nucleotide sequencesubstantially free of the sequences which would adjoin it in thewild-type virus associated with the sequence, the sequence beingselected from the group consisting of:

(a) the HVT homologue of the HSV gB gene,

(b) the HVT homologue of the HSV gC gene,

(c) the HVT homologue of the HSV gH gene,

(d) the TK gene of ILTV,

(e) the ILTV homologue of the HSV gH gene,

(f) ORF2 of ILTV,

(g) ORF3 of ILTV,

(h) the ribonucleotide reductase (large subunit) gene of ILTV,

(i) the ribonucleotide reductase (large subunit) gene of HVT,

(j) the ribonucleotide reductase (large subunit) gene of MDV,

(k) the ribonucleotide reductase (large subunit) gene of MDV,

(l) the HVT homologue of the immediate early gene IE-175 of HSV-I, and

(m) the HVT homologue of the immediate early gene IE-68 of HSV-I,

and minor variations thereof.

Each of sequences (a) to (m) may be associated with further elementssuch as suitable stop and start signals and other 5′ and 3′ non-codingsequences, including promoters, enabling expression of the sequence.Such further elements may be those associated with the sequence in itsnaturally-occurring state or may be heterologous to that sequence.

In particular the promoter may be one associated with one of thesequences (l) and (m) above.

The term “minor variations thereof” is intended to include changes inthe nucleotide sequence which do not affect its essential nature, forexample minor substitutions of nucleo-tides for one another. In the caseof sequences which are intended for insertion into a vector to encode anantigen, the “essential nature” of the sequence refers to the(glyco)protein encoded. Conservative changes in the nucleotide sequencewhich give rise to the same antigen will clearly be included, as willchances which cause conservative alterations in the amino acid sequencewhich do not affect adversely the antigenic nature of the antigen, inparticular, antigenic portions of the antigen sequences may be usedalone, for example the regions corresponding to nucleotides 273-320 or867-926 of HVT gH and minor variations thereof. These sequences and thepeptides encoded thereby form a further aspect of the invention. In thecase of a sequence which is an insertion site, it is necessary only thatthe sequence should be non-essential for the infectivity, andreplication of the virus and have sufficient homology with the definedsequence to enable recombination to occur. Thus an insertion of onenucleotide into the sequence could completely change the reading framefrom then on in a downstream direction. In the case of anantigen-encoding sequence this would usually alter the amino acidsequence undesirably (depending on where the frameshift occurred), butin the case of an insertion site, the degree of homology would be almostthe same, thereby allowing recombination to take place with almost thesame ease.

Generally speaking, in an insertion site, if a nucleotide homology of atleast 75% is present, the sequence is regarded as a “minor variation”.Preferably, the sequence is at least 80, 85, 90, 95 or 99% homologous.

It will be appreciated that such degrees of homology relate tosubstantially the entire portion of each sequence (a) to (m) definedabove. Shorter sequences may be used as probes in the identification orisolation of such longer sequences, but in this case the degree ofhomology will in general need to be greater in order to ensure accuratehybridisation.

Thus, a further aspect of the invention provides sub-sequences of atleast 13 nucleotides having at least 90% (preferably 95%, 99% or 100%)homology with at least one portion of any of the said sequences (a) to(m) above.

In the above list, sequences (a) to (c), (e), (f), (l) and (m) areuseful for expressing viral antigens. Sequences (b), (d) and (g) to (k)and, in addition, the TK region of MDV are useful as non-essential sitessuitable for insertion of antigen-expressing genes. Thus, sequence (b)is useful for both functions.

The sequences may readily be isolated from naturally-occurring ILTV, HVTand MDV viruses, using the sequence information given herein andstandard techniques, for example involving the preparation ofoligonucleotide probles and use thereof to hybridise to thenaturally-occurring DNA.

Antigenic ILTV and HVT sequences, i.e. sequences (a) to (c), (e), (f),(l) and (m) above, may be expressed in any suitable host and, inparticular, in HVT or MDV. Suitable non-essential sites for insertion ofone ILTV sequence include the MDV homologue of the HSV gC gene, the HVThomologue of the HSV gC gene, the TK gene of HVT or MDV, theribonucleotide reductase (large subunit) gene of HVT or MDV and theribonucleotide reductase (small subunit) gene of MDV.

A second aspect of the invention provides insertional or deletionalmutants of MDV, HVT and ILTV as follows:

(i) for HVT, a mutation in the region homologous to the HSV gC gene orin the ribonucleotide reductase gene or the TK gene,

(ii) for MDV, a. mutation in the region homologous to the HSV gC geneor, in the ribonucleotide reductase (small subunit) gene or in theribonucleotide reductase (large subunit) gene,

(iii) for ILTV, a mutation in the TK gene, ORF3 or the ribonucleotidereductase (large subunit) gene.

Each mutation may be in the coding or non-coding sequences of theregions identified.

Such mutant forms of HVT, MDV and ILTV may be used as, or created in thecourse of preparing, viral vectors for heterologous antigen-encodingsequences, or indeed as vectors for any other sequence which one wishesto express in a fowl in which the vector will replicate. Such sequencesinclude, but are not limited to, (a) to (c), (e), (f), (l) and (m).

By “heterologous”, we mean that the antigen-expressing sequence has notpreviously been found in the same place in relation to the remainder ofthe viral genome. For example, an antigen-expressing gene might beisolated from a virulent strain of ILTV and inserted into the TK regionof a less virulent strain of ILTV; this insertion would be regarded as“heterologous” if it did not result in a naturally-occurring virus.

The heterologous sequence may alternatively be one coding for an antigenassociated with any one of the following diseases: avianencephalomyelitis (epidemic tremor), avian influenza (fowl plague),avian leukosis, avian paramyxoviruses other than Newcastle disease (PMV2to PMV7), avian reovirus diseases (enteric disease, tenosynovitis),chicken anaemia (caused by chicken anaemia agent), coccidiosis, egg dropsyndrome (EDS76), fowl pox, infectious bronchitis, infectious bursaldisease (Gumboro), inclusion body hepatitis (adenovirus),lymphoproliferative disease of turkeys, Newcastle disease,reticuloendotheliosis in chickens, reticuloendotheliosis in turkeys,rotavirus enteritis, turkey haemorrhagic enteritis and turkeyrhinotracheitis. The sequence may alternatively encode paramyosin (amuscle protein common to all invertebrate parasites) or an antigenicpart thereof, somatostatin or a growth-promoting part thereof or animmune regulator.

The vectors in accordance with the invention may provide multivalentvaccine protection. For example, a vaccine comprising ILTV carrying anMDV antigen coding sequence Would be expected to protect against ILT andMarek's Disease.

Furthermore, the mutant ILTV viruses themselves are potentially usefulin vaccines as attenuated viruses, without necessarily having aheterologous sequence inserted.

A convenient process for preparing the deletional or insertional mutantsof the second aspect of the invention comprises simply introducing intoa suitable cell, for example by co-transfection, a deletional orinsertional mutant version of the appropriate region (for example, theTK region) and either whole viral DNA or a whole virus (for example thewild-type virus). The naked DNA of such viruses has been found to beinfectious, provided that it has not been sheared. A calcium phosphateprecipitate of the DNA is generally advantageous. Suitable cells includechicken embryo fibroblasts, chicken kidney cells and duck embryofibroblasts, all preferably grown in sub-confluent monolayers in Petridishes.

The transfected DNA and the whole viral DNA will then recombine withone-another in the infected cells by homologous recombination and thedesired recombinants can be screened for, for example by the detectionof hybridisation to suitable probes or by an immunoassay using suitableantibodies to the gene product of the region in question.

For homologous recombination to take place, the viral DNA mustreplicate. At present, no cell-free replication system for MDV, HVT orILTV is known. However, if such a system becomes available, then theprocess of the invention could be operated therein. The environment inwhich the replication and recombination occur is not critical.

The ILTV and HVT regions which were identified above as beingresponsible for encoding immunologically useful viral antigens can beinserted into suitable vectors, for example into HVT or into othervectors such as fowlpoxvirus, bacteria or fungi. In the case of viralvectors, especially herpesvirus vectors and poxvirus vectors, suchinsertion can be achieved by recombination between the antigen-encodingsequence, flanked by suitable non-essential sequences, and the vector'sgenome in a suitable host cell as described above. A promoter which isendogenous to the host will usually be used to control expression of theheterologous (viral antigen-encoding) sequence. In the case of bacteriaand fungi, the antigen-encoding sequence may be inserted using known oryet-to-be-discovered techniques of DNA manipulation. A non-pathogenicstrain of Salmonella may be used as such a host. The heterologoussequence may be inserted into the host's genome or be carried on anindependently-replicating plasmid.

The flanking sequences which are used may comprise all, virtually all orless of the region into which the heteroloaous sequence is to beinserted. If all the region is employed, then the sequence of thatregion will clearly still be present in the resulting virus, but thefunction of that region will have been deleted. If less than the wholeregion is used as flanking sequences, then the result will be astructural as well as functional deletion. Either approach may be used.

Thus, the construction of deletional or insertional mutants of ILTV canyield improved vaccines. Alternatively, the expression of ILTVglycoproteins or other ILTV proteins engineered into HVT, fowl pox orother vectors can constitute effective vaccines.

To prepare a vaccine in which HVT, MDV or ILTV is the virus or vector,the virus is grown in suitable cells such as chick embryo fibroblasts ina standard culture medium which as 199 medium (Wellcome or FlowLaboratories) for 3 to 4 days at about 37° C. The cells are harvested byscraping from the surface of the culture or by trypsinisation andsuspended in medium containing 1 mM EDTA or 10% dimethyl sulphoxide andin either case 4% calf serum before storage in liquid nitrogen in sealedampoules.

For vaccination. typically. day-old chicks are injected intramuscularlywith about 1,000 plaque-forming units. Immunity follows within a fewdays.

It should be noted that MDV and HVT are cell-associated viruses and areinfectious only when present in cells. Thus, a vaccine based on suchviruses will always include suitable infected cells.

The vaccines of the invention may be used to protect any fowlsusceptible to ILTV or HTV, including commercially-reared poultry suchas chickens. turkeys, ducks and quail.

Preferred aspects of the invention will now be described by way ofexample and with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of the MDV genome showing in part the BamH1 sitedistribution and the location of the RB and TK genes;

FIG. 2 (Parts A-R) shows the nucleotide sequence of the gB gene of theRB1B strain of MDV, with the numbering referring to the MDV nucleotides.the sequence of part of the HVT gB gene shown under the line. homologiesindicated by vertical bars. and amino acid differences between MDV gBand HVT gB shown above the line:

FIG. 3 is a map of the HVT genome showing the positions of the gHthatched), TK (solid black) and major capsid protein (MCP, dotted)genes, with HindIII sites shown as “H”:

FIG. 4 (Parts A-H) shows the nucleotide sequence of most of the HVT gHgene, with the corresponding amino acid sequence shown above the line;

FIG. 5 (Parts A-J) shows the nucleotide sequence of the HVT TK gene,with the numbering referring to the HVT nucleotides. the sequence ofpart of the MDV TK gene shown under the line, homologies indicated byvertical bars and amino acid differences between MDV TK and HVT TK shownabove the line;

FIG. 6 (Parts A-F) shows the nucleotide sequence of the gC gene of theRBIB strain of MDV, with corresponding amino acids shown above the line;

FIG. 7 (Parts A-K) shows the nucleotide and predicted amino acidsequence of a 5400 base pair region of the ILTV genome containing the TKgene cluster. Amino acid sequences predicted for the products of themajor open reading frames (ORFs) are indicated in the single letter codebelow the sequence for the strand and above the sequence for thecomplementary strand. The locations of potential ‘TATA’ boxes areunderlined. ORF 4 is the ILT TK gene sequence;

FIG. 8 is a representation of the gene organisation in the TK-containingpart of the ILTV genome. Overlapping pUC 13 plasmid clones containingthe EcoR1 (pILEc1) and BgIII (pILBg2) generated fragments of ILTV DNAare indicated. Open reading frames (ORFs) are depicted as open boxeswith the direction of transcription indicated by the arrow:

FIG. 9 shows part of the nucleotide sequence of the ILTV gB gene;

FIG. 10 shows part of the nucleotide sequence of the ILTV ribonucleotidereductase (large subunit):

FIG. 11 shows part of the nucleotide sequence of the HVT homologue ofthe VZV62/HSV-1 IE 175 gene:

FIG. 12 shows Dart of the nucleotide sequence of the HVT ribonucleotidereductase (large subunit) gene:

FIG. 13 (Parts A-B) shows part of the nucleotide sequence of the MDVribonucleotide reductase (large subunit) gene;

FIG. 14 shows part of the nucleotide sequence of MDV homologue ofribonucleotide reductase (small subunit) gene;

FIG. 15 shows part of the nucleotide sequence of the MDV homologue ofthe HSV-1 IE-175 gene;

FIG. 16 shows part of the MDV homologue of the HSV-1 IE-68 gene:

FIG. 17 is a schematic representation of homologous recombination at anon-essential region of a viral genome and a homologous region of DNAcloned within a plasmid vector; and

FIG. 18 is a map of plasmid pILBg2, showing restriction sites and thelocations of the TK gene and ORFs 3 and 5.

EXAMPLES

General Approaches

Selected short sequences of the avian herpesviruses cloned in thebacteriophage vector M13 were used as probes to identify longerfragments that might contain the entire genes of interest. This wasachieved by Southern blot hybridization of restriction fragments. Fulldetails are given below.

Virus Strains. The ‘highly oncogenic’ strain RB1B of MDV [Schat, K. A.et al (1982) Avian Pathol. 11, 593-605] was obtained from Professor B.Calnek, Cornell University, Ithaca, U.S.A. The virus received has beenplaque purified in chicken kidney vells in tissue culture. It waspassaged twice in SPF RIR chickens and 4 times in chick embryofibroblasts (CEF). Its ‘highly oncogenic’ nature was demonstrated by ahigh incidence of gross tumours when inoculated in genetically resistantN-line chickens.

The FC126 strain of HVT [Witter, R. L. et al (1970) Am. J. Vet. Res. 31,525-538], obtained from the Wellcome Research Laboratories, Beckenham,Kent, had been passaged 14 times in CEF. It was subsequently grown induck embryo fibroblasts (DEF) and CEF in our laboratory. It was thenplaque-purified and grown further in CEF. Viral DNA used for cloning inthe present work was extracted from virus that had been passed 29 timessince the original isolation.

The Thorne strain of ILTV was passaged twice in eggs, once in chickenkidney cells (CKC) and plaque-purified three times in CKC.

Tissue culture. CEF were grown in roller bottles in 199 medium(Wellcome), supplemented with penicillin, streptomycin, Fungizone (Regd,T. M.) and calf serum as described previously [Ross, L. J. N. et al(1975) J. gen. Virol. 28, 37-47].

CKC were grown in 10 cm Petri dishes [Churchill, A. E. and Biggs P. M.,(1967) Nature, 215, 528-530].

Isolation of MDV DNA. Cell associated RB1B was inoculated onto confluentmonolayers of CEF in roller bottles at a multiplicity of infection ofapproximately 0.001 plaque-forming units (pfu) per cell, and thecultures were incubated at 37° C. After 3 days, the medium was discardedand replaced with fresh 199 medium containing 2% calf serum. Cells wereharvested for virus purification after 2 to 3 days when cytopathiceffect was extensive. Virus was obtained by rate zonal centrifugation ofthe cytoplasmic fraction of infected cells [Lee, Y. S. et al (1980) J.gen. Virol. 51, 245-253]. Viral DNA was extracted by treating purifiedvirus with sarcosyl, proteinase K and Tris buffer pH 9 overnight at 37°C. and purified by rate zonal centrifugation in glycerol gradients asdescribed previously (Lee et al, 1980). High molecular weight viral DNAwas precipitated with ethanol and resuspended in 10 mM Tris pH 7.5 im 1mM EDTA (TE).

Isolation of ILTV DNA. (a) Infected CKC were harvested 2-3 days afterinoculation, washed in PBS, and resuspended in ice-cold TE by vortexing.Cells were lysed by addition of the non-ionic detergent NP40 (final 1%)vortexing and incubation on ice for 15 min. After treatment with RNAse,the preparation was centrifuged at 2000 rpm for 5 min in a bench topcentrifuge (Centaur). The supernatant was collected and incubated at 37°C. for 30 min in the presence of SDS (final 1%) and proteinase K (final0.5 mg/ml). The mixture was extracted twice with phenol-chloroform andonce with chloroform and the DNA was then precipitated with ethanol and1/10 vol of 3M sodium acetate.

(b) Viral DNA was also isolated from the media of virally infected cellsin the following way. The media of infected cells were harvested at 2-3days post infection and centrifuged at 3000 for 5 mins at 4° C. rpm in abench centrifuge. The supernatant was collected and centrifuged at 19Krpm in an ultracentrifuge (Sorvall) for 1 hr at 4° C. The viral pelletwas resuspended in TE, digested with RNAse A, then disrupted with SDSand proteinase K as described above. Finally, DNA was extracted from thedisrupted-virus as described above.

Cloning of MDV DNA. One fg of MDV DNA was cut with the restrictionenzyme BamH1 and ligated to BamH1-cut, dephosphorylated pUC13 DNA(Pharmacia). Competent E. coli strain TG1 cells were transformedaccording to standard procedures [Hanahan, D. (1983) J. Mol. Biol. 166,557-580] and were grown in the presence of ampicillin and X-gal. Whitecolonies were picked and tested for the presence or MDV inserts byhybridization to nick-translated MDV DNA [Grunstein M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3961]. Positive colonieswere cultured in small volume and plasmid DNA isolated by the procedureof Holmes, D. S. and Quigley, M. [(1981) Anal. Biochem. 114, 193-297].The size of the inserts was determined by electrophoresis of BamH1digests of the recombinant DNA in agarose gels. Plasmids containing MDVinserts ranging from less than 1 to 18 Kbp were obtained.

Cloning of ILTV DNA. EcoR1 and Bg1II libraries of ILTV DNA were obtainedby cloning digests of viral DNA in pUC13 as described above.

Random sequencing of viral DNA. Sonicated fragments of viral DNA werecloned into SmaI-cut, dephosphorylated M13.mp10 (Amersham InternationalPLC) and plaques containing MDV inserts were identified by hybridizationto MDV DNA. The sequence was determined by the dideoxy method [Sanger,F. et al (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467] using ³ ⁵SdATP).

The same procedure was used to sequence cloned fragments of MDV, HVT andILTV DNA except that plaques were identified by hybridization tolabelled insert so as to avoid colonies containing pUC13 fragments.

Example 1

gB Gene of MDV

An M13 clone of HVT homologous to the gB gene of VZV and HSV hybridizedto BamH1 fragment I3 of MDV (see FIG. 1). Sequencing of this fragmentobtained from a BamH1 library of the RB1B strain of MDV showed that twothirds of the gene, starting with the NH₂ terminus, was contained withinI3. The remainder of the gene was identified in the adjacent restrictionfragment K3. FIG. 1 shows the map position of the gene which is 2.6Kbplong. Its mRNA has been estimated to be approximately 2.8 Kb. Thetranslated protein is 865 amino acids long (FIG. 2). This includesapproximately 20 amino acids which may be part of a signal sequencedomain. The primary translated sequence of MDV gB has a few features incommon with gB of other herpes viruses such as the alignment of cysteineresidues and the presence of hydrophobic sequences which are presumablycapable of spanning a lipid bilayer [Pellet, P. E. et al (1985), J.Virol. 53, 243-253]. However, MDV gB has only 48% amino acid similaritywith gB of HSV and has many unique features such as the insertion of 23amino acids (residues 1851-1920, FIG. 2) and the presence of extra siteswith glycosylation potential. Comparison of the sequence of MDV gB withlimited sequence data (702 bases) available for HVT gB (FIG. 2) hasshown 76.9% nucleic acid similarity and 87.1% amino acid similaritybetween these two glycoproteins. Amino acid substitutions in HVT gBcompared to LMDV gB were particularly marked in a region (residues1323-1433) equivalent to a domain of HSV gB associated with virusneutralization [Pellet P. E. et al (1985) as above]. Amino acidsubstitutions between MDV and HVT gB were also noted in other regions ofunknown function.

Example 2

gH gene of HVT and gH Gene of MDV

An M13 clone of HVT containing sequences homologous to HSV gH wasisolated during our earlier work on gene identification and mapping(Buckmaster et al (1988) as above). This clone, when used as a probe,hybridized to a 6Kbp HindIII fragment of HVT (FIG. 3). Sequencingrevealed that this fragment contained approximately one quarter of thegH gene including the carboxy terminus. The adjacent HindIII fragment(3.2 Kbp) containing the remainder of the gH gene was identified byhybridization using a cloned HpaI fragment of HVT which overlapped theHindIII site. FIG. 4 shows the sequence of the coding region of the gHgene of HVT (2.3 Kbp) and flanking sequences. The % amino acid identitybetween the gH gene of HVT and its homologue in HSV1, VZV and EBV wasonly 20, 24 and 20 respectively (estimated from maximised amino acidoverlaps of 630, 644 and 153 respectively).

Example 3

TK Gene of HVT and TK Gene of MDV

The whole coding region of the TK gene of HVT (1053 bp) was containedwithin the 3.2 Kbp HindIII fragment described above (FIG. 3). Thesequence of the entire gene and flanking regions is shown in FIG. 5.Similarly the whole of the MDV TK gene is contained within the 3.6 KbpBamH1 K2 fragment of MDV (FIG. 1). The sequence of MDV TK genedetermined so far is shown in FIG. 5. Comparison of the LMDV and HVT TKsequences indicates that the two genes have approximately 60% amino acididentity (estimated from 276 amino acid overlap). By contrast, the %amino acid identities between the TK gene of HVT and the TK genes of HSV1, VZV and EBV are only 30, 27 and 24 respectively (estimated from aminoacid overlaps of 320, 332 and 193 respectively). The predicted aminoacid sequences of HVT and MDV TK show characteristic ATP and/or CTPbinding site motifs described for a number of virus and eukaryoticproteins that are associated with phosphorylation (Gentry, G. A. (1985)Proc. Natl. Acad. Sci. U.S.A. 82, 6815-6819). These conserved sequencesare examples of useful sites for insertion and expression of foreigngenes and for producing TK− deletion mutants.

Example 4

A Antigen Gene of MDV (gP57-65) (gC Homologue)

The A antigen gene is of interest in vaccine development both as animmunogen (it encodes a major glycopolypeptide product) and also becausewe have identified it as the homologue of HSV gC, a potentialnon-essential region. The A antigen gene was mapped within the BamH1 Bfragment of MDV (Isfort et al 1987), and the nucleotide sequencedetermined for the GA strain of MDV (Coussens and Velicer, AbstractOP18.51, VII International Congress of Virology, 9-14 August, (1987)Edmonton, Canada; J. Virol. 62, 2373-2379). During the random sequencingstudies described earlier (Buckmaster et al 1988), we identified an M13clone (No. 130) which came from the A antigen gene. This clone was thenused to identify a 2.3 Kbp EcoR1/PvuII fragment from the RB1B strain ofMDV containing the A antigen. This fragment was cloned into aSmaI/EcoR1cleaved pUC13 vector by standard protocols. One plasmid(pMB419) was sequenced by the M13 dideoxynucleotide method. The sequenceof the MDV RB1B A antigen and the predicted amino acid sequence of theprotein are presented in FIG. 6. The A antigen regions of MDV and HTVare non-essential genes and they can therefore be used as sites in MDVand HVT into which other genes can be inserted into the virus byhomologous recombination. Several lines of evidence support this asoutlined below.

1) During our study we isolated and sequenced another RB1B A antigenclone. This had one extra T residue in the string of T's 45 bases 3′ tothe A antigen ATG codon. This extra T would cause a frameshift whichwould make it impossible for the gene to encode functional A antigen. Asit is probable that this gene was cloned from a replicating MDV, theresults suggest that the A antigen is non-essential to the virus.

2) On conducting a similarity search it became clear that the MDV Aantigen gene is the homologue of HSV gC and PRV gpIII glycoproteins.Both of these homologous genes are known to be non-essential [for theHSV homologue, see Rosenthal et al (1987) J. Virol. 61, 2438-2447].

3) Strains of MDV lacking A antigen as judged by agar gel diffusiontests [Churchill, A. E. et al (1969) J. gen. Virol. 4, 557-564] orproducing low levels using the more sensitive 2Dradio-immunoprecipitation [van Zaane, D. et al (1982) Virology 121,133-146] have been reported.

Furthermore, in view of the fact that the A antigen is a major secretedglycoprotein, it may be a particularly suitable location for thepresentation of foreign epitopes within the A antigen as soluble,secreted proteins. This may. be achieved by cloning oligonucleotidesencoding these epitopes in frame within the A antigen gene.

STRATEGIES FOR INTRODUCING GENES INTO HVT AND ILTV VECTORS

Two possibilities can be envisaged. 1) Insertion into non-essentialgenes of the vector. 2) Substitution of foreign gene for correspondinggene of the vector. This would be possible only in regions which alreadyhave substantial homology as may be the case between some genes of MDVand HVT.

Example 5

Insertion into Non-essential Genes of HVT, ILTV or MDV

(a) Insertion at the TK locus of the vector.

1) HVT, ILTV or MDV may be used as vectors for insertion and expressionof avian herpesvirus genes. In particular gB, gD, gH or gC of RB1B MDVmay be inserted into ILTV. Also gB and BS-17 of ILTV may be insertedinto HVT or MDV. One may use the promoter associated with the insertedgene or use heterologous promoters, including those of a different classof genes (for example the immediate early promoter to optimiseexpression of gB).

2) ILTV may be used as a general vector for the insertion and expressionof genes unrelated to avian herpes viruses and likely to requiremanipulation of promoters for optimal expression.

The procedure to be used for gene insertion is substantially asdescribed previously for the insertion of hepatitis antigen in HSV [Shihet al, 1984 as above].

MDV and HVT DNA obtained as described above is infectious provided thatprecautions are taken not to shear the DNA during extraction. Calciumphosphate precipitates of viral DNA prepared as described by Stow andWilkie [(1976) J. gen. Virol. 33, 477] were added to sub-confluentmonolayers of CEF. After absorption for 1 h at 37° C., culture mediumwas added and cultures were incubated for 1 or 2 days until confluent.Monolayers were then trypsinised, replated (1:1 or 1:2) in 199 medium(Wellcome) containing 2 to 4% calf serum and incubated at 37° C. untilplaques developed, usually after 4 to 5 days. Approximately 200 plaquesmay be obtained per μg of HVT DNA and approximately 50 per μg of MDVDNA.

Restriction enzyme sites than could be used for the insertion of foreignantigens into the TK of HVT strain Fc-126 include: BanII, Bsp1286,DraIII, EcoRI, HincII, HpaI, NheI and NspbII.

Some of these enzymes also have sites in the plasmid vector into whichthe virus DNA fragments are cloned. Thus in order to linearize the cloneDNA without also cutting within the vector, partial digests may becarried out.

None of the above enzymes should cause any disruption to flanking gene,HSV-1 homologues of which are known to play an important role in virusmultiplication.

For homologous recombination and isolation of recombinant virus, genesof interest are inserted within non-essential genes such as TK or gC andco-transfected with wild-type viral DNA at molar ratios ranging from10:1 to 2:1 as described above. Alternatively, intact wild-type virusmay be used for co-infection.

Virus recombination may be detected by ‘plaque lifts’ which involvetransfer of infected cells and released virus which have adhered to theagar overlay to nitrocellulose and hybridization of the denatured DNAreleased from the cells and virus to suitable probes as described byVillareal, L. et al (1977) Science 196, 183-185. Virus which hybridizesto. the probe may be recovered from the monolayer.

A similar procedure may be used to isolate recombinant virus whichexpressed epitopes of interest. In this instance the nitrocellulose“plaque lifts” are treated with antibody and the presence of boundantibody revealed using a suitable detection system such as labelledprotein A or phosphatase conjugated anti-globulin antibody.

The gene of interest with appropriate promoters is first inserted withinthe cloned TK gene (FIG. 7). The recombinant DNA is then co-transfectedwith infectious DNA of the vector in chick embryo fibroblasts or chickenkidney cells and TK− virus may be selected by growth in mediumcontaining-acyclovir [Ross, N. (1985) as above] or FMAU [Schat, K. A. etal (1984) Antiviral Research 4, 159-70]. Alternatively, or in addition,plaques are screened for the presence of the gene of interest using‘plaque lifts’ on nitrocellulose and hybridization to any relevantlabelled probe. Plaques are also screened for expression of the epitopesof interest using monoclonal antibodies or antipeptide antibodies.

The main advantage of this strategy is that the selection procedureincreases the chances of obtaining virus recombinants containing thegene of interest. It also offers the opportunity of using differentpromoters for optimum expression. Thus the use of an immediate earlypromoter may allow expression in latently infected cells.

(b) Insertion at the gC Locus of the Vector.

Since the A antigen (HVT and MDV homologues of HSV gC) is not essentialfor virus growth in vivo and in vitro (see section on gC above) it is apotentially useful site for the insertion and expression of foreigngenes. Moreover, since it is one of the most abundant antigens and isexcreted, it may be particularly useful for enhancing the immunogenicproperties of foreign proteins. The isolation of virus recombinants atthis locus may be achieved by first inserting at least part of the geneof interest in frame within the gC gene and then co-transfecting withinfectious viral DNA. Screening of virus plaques with sequence specificprobes or with specific antibody allows the isolation of recombinants.

Example 6

Substitution or ILTV Genes for Their Homologues in HVT

Substitution may be achieved by co-transfection of cloned ILTV sequencesand infectious HVT DNA as described in Example 5. Substitution of genesderived from ILTV for their counterparts in HVT may be effected.

Recombinants expressing ILTV sequences and epitopes may be detectedusing ILTV-specific monoclonal antibodies or anti-peptide antibodiesraised against unique ILTV sequences as described above.

The advantage of this procedure is that it is relatively simple and doesnot require manipulation of promoters. However, it may be limited togenes which share substantial homology.

Example 7

Strategies for Obtaining TK− Mutants of ILTV

Deletion mutants. Deletions may be introduced within any suitable partof the gene, for example the domains of the gene that are required forits function as a phosphorylating enzyme such as ATP and CTP bindingsites. This may be achieved by restriction enzyme digestion, for examplewith SnaB1 or Bc1I, and religation of appropriate fragments followed byco-transfection with infectious viral DNA or transfection intovirally-infected cells. Reference may be made to FIGS. 7 and 8, and tothe map of plasmid pILBg2 (FIG. 18), in choosing restriction enzymes andso on. TK− virus may be selected in the presence of acyclovir [Ross, N.(1985) as above] or FMAU [Schat, K. A. et al (1984) as above].Plaque-purified clones may then be tested for the absence of the deletedportion of the TK gene by hybridization.

The deletion mutants of ILTV may be used themselves as attenuatedviruses for vaccine preparation, or may have sequences for heterologousantigens inserted.

Insertional mutants. A functional β-galactosidase gene under the controlof a herpesvirus promoter or any other suitable sequence or a singlebase is first introduced in a domain of the TK gene which is essentialfor TK activity. The recombinant DNA is then co-transfected withinfectious viral DNA or transfected into virally-infected cells to allowhomologous recombination to occur. Selection in the presence of acyloviror FMAU will yield TK− insertional mutants. If a β-galactosidase gene isintroduced, mutants can be detected by the production of blue plaques inthe presence of X-gal.

The TK gene and surrounding sequences may be subcloned into anothersuitable vector if necessary.

Example 8

Insertion of MDV RB1B gB Gene into HVT

(Not within the scope of the invention, but illustrates an analogoustechnique).

The HVT TK gene is cloned in the plasmid vector pUC13 to generate aplasmid, which may be termed pTK1B. This plasmid is linearised with, forexample, the restriction endonuclease Rsr II which cleaves the plasmidonly within the TK gene (nucleotide position 197 in FIG. 5, enzymerecognition sequence CGGACCG). The “sticky” ends thus generated are endrepaired by standard techniques (see “Molecular Cloning: a LaboratoryManual”, ed. Maniatis T., Fritsch E. F., and Sambrook J. Cold SpringHarbor Laboratory 1982).

The RB1B gB was originally cloned on two plasmids which were termedRBIB-BamH1-I₃ and RB1B-BamH1-K₃. (Note I₃ had lost one BamH1 site duringcloning.) To generate a complete gB copy on one plasmid, both plasmidswere cleaved with BamH1 and the fragments ligated. However, the completegB gene was later obtained independently on an EcoRI/SalI fragment. Rosset al, J. gen. Virol (1989) 70, 1789-1804 provides further informationregarding the manipulation of viral genes. Recombinants containing thedesired configuration can be identified by restriction enzyme analysisof plasmid DNA's.

The recombinant plasmid is then cleaved with EcoR1, the ends arerepaired and the plasmid is cloned into PTK1B prepared as above. Therecombinant plasmid is then introduced into cells containing HVT virus(viral DNA) and homologous recombination will introduce the gB gene intothe TK gene. HVT viral recombinants can be selected with acyclovir orFMAU or alternatively detected with labelled gB probes.

Example 9

RB1B gC (A antigen) Gene into HVT

Blunt ended PTH1 B is prepared as in example 8. The RB1B gC is cleavedfrom the plasmid pMB419 (Example 4) with the restriction endonucleasesEcoR1 and HindIII (site within the pUC13 polylinker). The sticky endsgenerated are again end-repaired by standard protocols. The end-repairedgC fragment is then cloned into the linearized end-repaired pTK1B as inExample 8. (The cloning can be verified by analysis of the resultingclones with restriction enzymes, probing with radioactively labelledfragments, or DNA sequencing, or any combination of these).

The resulting plasmid with the RB1B gC gene cloned into the HVT TK genecan then be introduced into the HVT genome by transfecting the plasmidinto HVT-infected cells using calcium phosphate precipitation orelectro-poration. Homologous recombination, involving crossovers eitherside of the gC gene, between the HVT virus and the flanking sequences ofthe HVT TK plasmid will carry the RB1B gC gene into the HVT viralgenome. Viral recombinants can be selected for (as they are TK−) oridentified (eg by probing) as described above.

In analogous ways, the sequence information given above and in theFigures can be used to design cloning strategies for the insertion ofthese genes and others into the non-essential genes of the ILTVdescribed here or to generate combinations of antigen genes into ILTV.

What is claimed is:
 1. A recombinant ILTV synthetically modified by amutation in an ILTV gene such that the recombinant ILTV is attenuated asa result of said mutation.
 2. The ILTV according to claim 1, wherein themutation is in the TK, ORF3 or ribonucleotide reductase, large subunit,gene.
 3. The ILTV according to claim 1, synthetically modified by thepresence, in a non-essential region of the ILTV genome, of DNA notnaturally occurring in ILTV.